The focus of this proposal is to dissect the role of Protein Homeostasis in enterovirus replication, evolution and pathogenesis. The paradigm for these studies is that enteroviruses are highly dependent on the cellular chaperone and quality control (QC) machinery for their protein production and function. Furthermore, given the very high mutation rates of these viruses, we hypothesize that the chaperones and QC network is key to modulating virus diversity, evolution and pathogenesis. We propose to examine how host protein homeostasis machinery participates in enterovirus replication and modulates viral diversity and how viruses use this machinery to alleviate the detrimental effect of mutations that accumulate during replication. We have recently demonstrated that high mutation rate of enteroviruses is essential for their adaptability and pathogenesis. Thus, restricting viral diversity through the modulation of protein homeostasis machinery could provide powerful ways to attenuate virus pathogenesis. This proposal combines computational, cell biological, molecular and systems approaches to define the host protein homeostasis network involved in enteroviral replication and determine the role of this network to viral evolution, diversity, and pathogenesis. These new approaches are essential given the failure of traditional strategies to achieve any therapeutic intervention against enteroviruses (such as polio, coxsackie, and enterovirus 71) and it has the potential to set the basis for the prevention of current and emerging enteroviral diseases . This proposal describes a multidisciplinary and highly integrated approach that is designed to obtain this critically important information. We propose 3 Specific Projects and 2 Cores: Project 1: Host-enterovirus circuitry revealed by global analysis of cellular networks;Project 2: Role of cellular factors in enterovirus protein homeostasis and function;and Project 3: Role of protein homeostasis in enterovirus population diversity, evolution and pathogenesis. Core A: Administrative Core;and Core B: "High-throughput functional genomics and proteomics core. PUBLIC HEALTH RELEVANCE (provided by applicant): Enteroviruses have been associated with many clinically recognized, life-threatening syndromes. Virus evolution is at the core of virus drug resistant, immunological survey escape and pathogenesis. Understanding protein homeostasis will illuminate the rules that govern virus diversity, evolution and pathogenesis, and it may allow the development of safe and effective vaccines and anti-enterovirus drugs. PROJECT 1: Title: Unbiased Functional Characterization of Enterovirus-Host Interactions Project Leader: Krogan, Neven PROJECT 1 DESCRIPTION (provided by applicant): In this study, we aim to functionally interrogate host-pathogen relationships using three different enteroviruses (Poliovirus, EV71 and Coxsackievirus). To this end, we will use a variety of methods to systematically generate viral-host protein-protein and genetic interaction maps. The data generated using these initial, unbiased approaches will fuel more targeted, hypothesis-driven research in the subsequent projects. Although we intend to follow up on the most interesting, unanticipated connections we uncover, we will be closely monitoring for links to host factors involved in quality control processes, including chaperone function, protein ubiquitination and protein degradation, which will link this work to the work described in Projects 2 and 3. In collaboration with Sumit Chanda (Burnham Institute) and John Young (Salk Institute), we will utilize RNAi methodology to globally assess the genetic dependencies, both positive and negative, of host factors to the pathogenesis of the three enteroviruses (Aim 1). Next, to characterize the enterovirus-human protein-protein interactions, we intend to collaborate with Al Burlingame (UCSF) to employ a systematic affinity tag/purification-mass spectrometry approach to identify the viral-host protein complexes (Aim 2). We also intend to globally ascertain the effects of protein post-translational modifications upon infection using mass spectrometry (Aim 3). Finally, In Aim 4, we will utilize a suite of bioinformatic and visualization tools to integrate the datasets in a meaningful fashion so that specific hypotheses regarding quality control processes can be generated and tested in collaboration with Judith Frydman (Project 2) and Raul Andino (Project 3). This integrated approach will leverage the expertise from multiple groups, including PIs of the Technology Core (Andrej Sali and Joe DeRisi), so that novel host pathways that are hijacked during Infection can be identified and characterized. This information will hopefully lead to breakthroughs with anti-viral drugs and vaccines. PUBLIC HEALTH RELEVANCE (provided by applicant): Human enteroviruses infect millions of people worldwide each year, resulting in a wide range of clinical outcomes ranging from respiratory illness to meningitis. To gain insight into the pathogenesis of these viruses, we aim to analyze the functional, genetic and biochemical relationships between several members of this class of virus and host cells, which will reveal key human pathways that are being hijacked during infection. This information can ultimately be used to generate novel anti-viral drugs and vaccines.